Human Respiratory Syncytial Virus (RSV) is the leading cause of hospitalization for viral respiratory tract disease (e.g., bronchiolitis and pneumonia) in infants and young children worldwide, as well as a significant source of morbidity and mortality in immunocompromised adults and in the elderly (see, e.g., Shay et al. (1999) “Bronchiolitis-associated hospitalizations among US children, 1980-1996” JAMA 282:1440-1446, Falsey et al. (1995) “Respiratory syncytial virus and influenza A infections in the hospitalized elderly” J Infect Dis 172:389-394, Falsey et al. (1992) “Viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings” J Am Geriatr Soc 40:115-119, Falsey and Walsh (1998) “Relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults” J Infect Dis 177:463-466, Hall et al. (1986) “Respiratory syncytial viral infection in children with compromised immune function” N Engl J Med 315:77-81, and Harrington et al. (1992) “An outbreak of respiratory syncytial virus in a bone marrow transplant center” J Infect Dis 165:987-993). To date, no vaccines have been approved which are able to prevent the diseases associated with RSV infection. RSV is an enveloped virus that has a single-stranded negative sense nonsegmented RNA genome, and it is classified in the Pneumovirus genus of the Paramyxoviridae family (Collins et al. (2001) Respiratory syncytial virus. pp. 1443-1485. In; Knipe and Howley (eds.) Fields Virology vol. 1. Lippincott, Williams and Wilkins, Philadelphia; Lamb and Kolakofsky (2001) Paramyxoviridae: the viruses and their replication pp. 1305-1340. In; Knipe and Howley (eds.) Fields Virology vol. 1. Lippincott, Williams and Wilkins, Philadelphia). Human RSV is classified into two subgroups, subgroups A and B, based on antigenic diversity and nucleotide sequence divergence. For example, the attachment protein G is most divergent and the fusion protein F is relatively conserved between the two subgroups.
Considerable progress has been made towards understanding the molecular biology of subgroup A RSV; however, much less information is available for subgroup B RSV. Most work to date has focused on subgroup A strains. For example, RSV strain A2 has been sequenced. The genome of the A2 strain RSV is 15,222 nt in length and contains 10 transcriptional units that encode 11 proteins (NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L). The genome is tightly bound by the N protein to form the nucleocapsid, which is the template for the viral RNA polymerase comprising the N, P and L proteins (Grosfeld et al. (1995) J. Virol. 69:5677-5686; Yu et al. (1995) J. Virol. 69:2412-2419). Each transcription unit is flanked by a highly conserved 10-nt gene-start (GS) signal, at which mRNA synthesis begins, and ends with a semiconserved 12- to 13-nt gene-end (GE) signal that directs polyadenylation and release of mRNAs (Harmon et al. (2001) J. Virol. 75:36-44; Kuo et al. (1996) J. Virol. 70:6892-6901). Transcription of RSV genes is sequential, and there is a gradient of decreasing mRNA synthesis due to transcription attenuation (Barik (1992) J. Virol. 66:6813-6818; Dickens et al. (1984) J. Virol. 52:364-369). The viral RNA polymerase must first terminate synthesis of the upstream message in order to initiate synthesis of the downstream mRNA.
The nucleocapsid protein (N), phosphoprotein (P), and large polymerase protein (L) constitute the minimal components for viral RNA replication and transcription in vitro (Grosfield et al. (1995) J. Virol. 69:5677-5686; Yu et al. (1995) J. Virol. 69:2412-2419). The N protein associates with the genomic RNA to form the nucleocapsid, which serves as the template for RNA synthesis. The L protein is a multifunctional protein that contains RNA-dependent RNA polymerase catalytic motifs and is also probably responsible for capping and polyadenylation of viral mRNAs. However, the L protein alone is not sufficient for the polymerase function; the P protein is also required. Transcription and replication of RSV RNA are also modulated by the M2-1, M2-2, NS1, and NS2 proteins that are unique to the pneumoviruses. M2-1 is a transcription antitermination (or elongation) factor required for processive RNA synthesis and transcription read-through at gene junctions, essential for RNA transcription and virus replication (Collins et al. (1996) “Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus” Proc Natl Acad Sci USA 93:81-85; Hardy and Wertz (2000) “The Cys3-His1 motif of the respiratory syncytial virus M2-1 protein is essential for protein function” J Virol 74:5880-5885; Tang et al. (2001) “Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability” J Virol 75:11328-11335; Collins et al. (2001) in D. M. Knipe et al. (eds.), Fields Virology, 4th ed. Lippincott, Philadelphia; Hardy et al. (1999) J. Virol. 73:170-176; and Hardy and Wertz (1998) J. Virol. 72:520-526). M2-2, though not essential for virus replication in tissue culture, is involved in the switch between viral RNA transcription and replication (Bermingham and Collins (1999) Proc. Natl. Acad. Sci. USA 96:11259-11264; Jin et al. (2000) J. Virol. 74:74-82). NS1 and NS2 have been shown to inhibit minigenome synthesis in vitro (Atreya et al. (1998) J. Virol. 72:1452-1461).
NS1, NS2, SH, M2-2 and G are accessory proteins that can be deleted from the RSV A2 strain without affecting virus viability (Bermingham and Collins (1999) Proc. Natl. Acad. Sci. USA 96:11259-11264; Jin et al. (2000) J. Virol. 74:74-82; Jin et al. (2000) “Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo” Virology 273:210-218; Bukreyev et al. (1997) “Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse” J Virol 71:8973-8982; Teng and Collins (1999) “Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein” J Virol 73:466-473; Teng et al. (2000) “Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees” J Virol 74:9317-9321; Karron et al. (1997) “Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant” Proc Natl Acad Sci USA 94:13961-13966). However, except for the SH deletion mutant, most of the gene deletion mutants do not replicate as well as the wild type RSV either in tissue culture or in animal hosts.
The G and F proteins are the two major surface antigens that elicit anti-RSV neutralizing antibodies to provide protective immunity against RSV infection and reinfection. High levels of circulating antibodies correlate with protection against RSV infections or reduction of disease severity (Crowe (1999) Microbiol. Immunol. 236:191-214). As noted, two antigenic RSV subgroups (A and B) have been recognized based on virus antigenic and sequence divergence (Anderson et al. (1985) J. Infect. Dis. 151:626-633; Mufson et al. (1985) J. Gen. Virol. 66:2111-2124). By using a reciprocal cross-neutralization assay, it has been determined that the F proteins between the two subgroups are 50% related and the G proteins are only 1-7% related (reviewed by Collins et al. (2001) “Respiratory syncytial virus” In: D. M. Knipe et al. (Ed) Fields Virology, pp. 1443-1485, Vol. 1, Lippincott Williams & Wilkins, Philadelphia). This antigenic diversity may be partly responsible for repeated RSV infection. The antigenic diversity of these two RSV subgroups enables viruses from both subgroups to circulate concurrently in a community, and the prevalence of each subgroup can alternate during successive years. Epidemic studies of RSV infection in children have suggested that naturally acquired infection elicits a relatively higher protection against disease caused by the homologous subgroup virus (McIntosh and Chanock (1990) “Respiratory syncytial virus” In: D. M. Knipe et al. (Ed) Second Edition Virology, pp. 1045-1072, Raven Press, Ltd., New York). The immunity induced by RSV infection is transient and subsequent reinfection can occur. However, RSV reinfection usually does not cause severe disease. An RSV vaccine is therefore typically targeted to provide protection against severe lower respiratory disease caused by RSV subgroup A and B viruses.
Efforts to produce a safe and effective RSV vaccine have focused on the administration of purified viral antigen or the development of live attenuated RSV for intranasal administration. For example, a formalin-inactivated virus vaccine not only failed to provide protection against RSV infection, but was shown to exacerbate symptoms during subsequent infection by the wild-type virus in infants (Kapikian et al. (1969) Am. J. Epidemiol. 89:405-421; Chin et al. (1969) Am. J. Epidemiol. 89:449-63). More recently, efforts have been aimed towards developing live attenuated temperature-sensitive mutants by chemical mutagenesis or cold passage of the wild-type RSV (Crowe et al. (1994) Vaccine 12:691-9). Typically, the virus candidates have been either underattenuated or overattenuated (Kim et al. (1973) Pediatrics 52:56-63; Wright et al. (1976) J. Pediatrics 88:931-6), and some of the candidates were genetically unstable, resulting in the loss of the attenuated phenotype (Hodges et al. (1974) Proc Soc. Exp. Bio. Med. 145:1158-64). To date, no live attenuated vaccine has been brought to market.
Characterization of additional strains of RSV, particularly from subgroup B, will assist in production of effective vaccines (e.g., regions of homology or identity between strains can indicate functionally conserved regions that can be targeted by mutagenesis). Although short regions of various subgroup B strains have been sequenced (e.g., B9320 protein G, SEQ ID NO:14, from GenBank accession number M73544; a B9320 intergenic region, SEQ ID NO:15, from GenBank accession number S75820; B9320 G and F gene start and end sequences, SEQ ID NOs:16-19, from Jin et al. (1998) Virology 251:206-214 and Cheng et al. (2001) Virology 283:59-68; and various B18537 coding and intergenic regions, GenBank accession numbers D00334, D00392-D00397, D00736, D01042, and M17213), only one subgroup B strain, strain B1, has been sequenced in its entirety (SEQ ID NO:13, from GenBank accession number AF013254).
Accordingly, this invention presents the complete polynucleotide sequence of human RSV subgroup B strain 9320. Polypeptides encoded by the B9320 genome are also provided, as are other benefits which will become apparent upon review of the disclosure.